Porphyrin compound, albumin inclusion compound thereof and artificial oxygen carrier

ABSTRACT

A porphyrin compound represented by general formula (A): 
                         
Also disclosed are a porphyrin metal complex-albumin inclusion compound having a porphyrin compound, in which M denotes Fe or Co, included in albumin, and an artificial oxygen carrier containing the porphyrin metal complex-albumin inclusion compound as an active component.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.10/799,128 filed on Mar. 12, 2004 now abandoned, which is based on, andclaims the benefit of priority from Japanese Patent Application No.2003-069760 filed Mar. 14, 2003, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention The present invention relates to a novelporphyrin compounds such as a porphyrin metal complex, an albumininclusion compound thereof, and an artificial oxygen carrier.

2. Description of the Related Art

Heme, i.e., a porphyrin iron (II) complex, constituting the activecenter of hemoglobin or myoglobin that plays the role of carrying andstoring oxygen within the living body, is capable of reversiblyadsorbing and desorbing molecular oxygen in response to oxygen partialpressure.

Many researches have been reported since 1970's in an effort to achievethe oxygen binding and dissociating capability similar to that performedby natural heme by using a synthetic porphyrin iron (II) complex. Asforerunner researches, for example, J. P. Collman, Acc. Chem. Res., 10,265 (1977) and F. Basolo, B. M. Hoffman, J. A., ibers, Acc. Chem. Res.,8, 384 (1975) can be cited. Particularly,5,10,15,20-tetrakis(α,α,α,α-pivalamidophenylporphyrin iron (II) complex(hereinafter referred to as “FeTpivPP complex”) is known as a porphyriniron (II) complex that is reported to be capable of forming an oxygencomplex that is stable under room temperature conditions (see J. P.Collman, et al., J. Am. Chem. Soc., 97, 1427 (1975). The FeTpivPPcomplex is capable of reversibly adsorbing and desorbing the oxygenmolecule at room temperature within an organic solvent such as benzene,toluene, dichloromethane, tetrahydrofuran or N,N-dimethylformamide if anexcess amount of an axial base such as 1-alkylimidazole or1-alkyl-2-methylimidazole is co-present. Also, if the complex isencapsulated in a bi-layer vesicle made of a phospholipid, the complexsimilarly exhibits an oxygen binding and dissociating capability evenunder the physiological conditions (aqueous system, pH 7.4, □ 40□) (seeE. Tsuchida et al., J. Chem. Soc., Dalton Trans., 1984, 1147 (1984)).

However, in order to allow the FeTpivPP complex to bind oxygenreversibly, it is necessary to add an excess molar amount of the axialbase compound from the outside as pointed out above. Some imidazolederivatives widely used as the axial base may produce a pharmaceuticaleffect and may exhibit a high toxicity in the living body. Also, in thecase of utilizing a phospholipid vesicle, the imidazole derivative thatis co-present in an excess amount may make the vesicle unstable. Toultimately decrease the addition amount of the axial base is tointroduce the imidazole derivative into the porphyrin molecule by thecovalent bond.

The research group including the present inventors have took theposition that a stable oxygen carrier can be provided without externallyadding the axial base, if an imidazole is covalently bonded, as a sidechain substituent, to the porphyrin iron (II) complex. Based on theparticular idea, a FeTpivPP analogue having a substituent in the2-position of the porphyrin ring have been accurately synthesized.Further, an inclusion compound having the FeTpivPP analogue included inthe phospholipid vesicle or the human serum albumin has been prepared,providing an artificial oxygen carrier capable of reversibly adsorbingand desorbing oxygen (see Japanese Patent Disclosure (Kokai) No.59-164791, Japanese Patent Disclosure No. 59-162924, Japanese PatentDisclosure No. 6-271577 and Japanese Patent Disclosure No. 8-301873).

However, most of the synthetic porphyrin iron (II) complexes capable offorming a stable oxygenated complex nowadays are tetraphenylporphyriniron complexes such as the FeTpivPP analogue. Derivatives having aproximal base covalently bonded to the protoporphyrin iron (II) complex,which constitutes the active center of hemoglobin or myoglobin withinthe living body, have been synthesized (see W. S. Brinigar, C. K. Chang,J. Geibel, T. G. Traylor, J. Am. Chem. Soc., 96, 5597 (1974)). However,these derivatives tends to readily form a μ-oxo dimer even within anorganic solvent such as N,N-dimethylformamide or toluene and, thus, thestability of its oxygenated complex is low, as compared to thetetraphenylporphyrin iron (II) complex. The group of the presentinventors have also synthesized a derivative in which an alkylimidazoleis covalently bonded to the protoporphyrin iron complex, and prepared anoxygenated complex in respect of the compound having the above-notedderivative included in albumin (see above mentioned Japanese PatentDisclosure No. 8-301873). However, the half-life period of itsoxygenated complex is not longer than one hour under 25□. It followsthat the oxygenated complex leaves room for further improvement in termsof the stability in the case of using the oxygenated complex as anartificial oxygen carrier.

Needless to say, the protoporphyrin iron complex derivative isadvantageous also in the case where the administration into the livingbody is taken into account. Protoporphyrin iron (III) that has been nolonger utilized in the living body is caught by heme oxidases so as tocleave the α-meso position of the porphyrin and thus is decomposed intobiliberdin so as to be used in the metabolic process. Since the hydrogenatom in the meso position is substituted with the phenyl ring in thetetraphenylporphyrin iron complex, the tetraphenylporphyrin iron complexmay not be decomposed in the metabolic mechanism.

Thus, in the case of considering the use of an aqueous dispersion of asynthetic porphyrin iron (II) complex as an artificial oxygen carrier,e.g., as a substitute for the erythrocyte, development of a moleculedesign and synthesis of a porphyrin iron derivative capable of formingan oxygenated complex having a higher stability and development of aninclusion compound having the particular porphyrin ion derivativeincluded therein are strongly desired.

BRIEF SUMMARY OF THE INVENTION

An object of the present invention is to provide a porphyrin compoundcapable of forming an oxygenated complex having a further improvedstability, an albumin inclusion compound thereof, and an artificialoxygen carrier.

As described above, the group of the present inventors have synthesizedand reported various porphyrin metal complexes having the oxygen bindingand dissociating function. Also, the group of the present inventors haveprovided the porphyrin metal complex-albumin inclusion compound havingthe porphyrin metal complex included in the serum albumin as anartificial oxygen carrier within water (see Japanese Patent DisclosureNo. 8-301873 referred to previously). Further, the group of the presentinventors have clarified that, where an intramolecular axiallycoordinated base is constituted by a histidine derivative, the stabilityof the oxygenated complex in the porphyrin metal complex-albumininclusion compound is improved, as compared to the case where theintramolecular axially coordinated base is constituted by animidazolylalkyl (see T. Komatsu, et al., Bioconjugate Chem., 13, 397(2002)). The present inventors have continued an extensive researchbased on the results of the research on the porphyrin metal complexcapable of coupling oxygen and the research on the porphyrin metalcomplex inclusion compound having the porphyrin metal complex includedtherein as an active site, accomplishing the present invention.

According to the present invention, there is provided a porphyrincompound represented by general formula (A):

where R¹ denotes a C₁-C₁₈ alkyloxy group, a C₁-C₁₈ alkylamino group, ora peptide having 1-6 α-amino acids and having a hydroxyl group, a benzyloxy group or a methoxy group at the C-terminal; R² denotes a residualgroup after removal of an amino group and a carboxyl group from anα-amino acid; R³ denotes a C₁-C₁₈ alkyloxy group, a C₁-C₁₈ alkylaminogroup, or a peptide having 1-6 α-amino acids and having a hydroxylgroup, a benzyloxy group or a methoxy group at the C-terminal; each R⁴and each R⁵ denote either a methyl group, or a hydrogen atom, a vinylgroup, an ethyl group, a 1-methoxyethyl group, a 1-bromoethyl group or aformyl group, wherein, where each R⁴ denotes a methyl group, each R⁵denotes a hydrogen atom, a vinyl group, an ethyl group, a 1-methoxyethylgroup, 1-bromoethyl group or a formyl group, and where each R⁴ denotes ahydrogen atom, a vinyl group, an ethyl group, a 1-methoxyethyl group, a1-bromoethyl group or a formyl group, each R⁵ denotes a methyl group; Mdenotes two hydrogen atoms bonded to the two pyrrole nitrogen atoms oran ion of a transition metal belonging to the fourth to fifth periods inthe Periodic Table; X⁻ denotes a halogen ion that is present where Mdenotes the transition metal ion; and n which denotes the number of X isthe number obtained by subtracting 2 from the valency of the transitionmetal ion.

Further, where M in general formula (A) denotes Fe or Co, the presentinvention provides a porphyrin metal complex-albumin inclusion compoundhaving the porphyrin metal complex included in albumin.

Furthermore, the present invention provides an artificial oxygen carriercontaining, as an active component, the porphyrin metal complex-albumininclusion compound of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in detail.

The porphyrin compound of the present invention is represented bygeneral formula (A) given above.

In general formula (A), R¹ denotes a C₁-C₁₈ alkyloxy group, a C₁-C₁₈alkylamino group, or a peptide having 1-6 α-amino acids and having ahydroxyl group, a benzyl oxy group or a methoxy group at the C-terminal.

R² denotes —(R)CH— group in an α-amino acid: H₂N(R)CHCOOH (for example,glycine (R═H), alanine (R=—CH₃), valine (R=—CH(CH₃)₂), leucine(R=—CH₂CH(CH₃)₂), and isoleucine (R=—CH(CH₃)C₂H₅), that is, a residualgroup after removal of an amino group and a carboxyl group from anα-amino acid.

R³ denotes a C₁-C₁₈ alkyloxy group, a C₁-C₁₈ alkylamino group, or apeptide having 1-6 α-amino acids and having a hydroxyl group, abenzyloxy group or a methoxy group at the C-terminal.

Each R⁴ and each R⁵ denote either a methyl group, or a hydrogen atom, avinyl group, an ethyl group, a 1-methoxy ethyl group, a 1-bromoethylgroup or a formyl group, wherein, where each R⁴ denotes a methyl group,each R⁵ denotes a hydrogen atom, a vinyl group, an ethyl group, a1-methoxyethyl group, 1-bromoethyl group or a formyl group, and whereeach R⁴ denotes a hydrogen atom, a vinyl group, an ethyl group, a1-methoxyethyl group, a 1-bromoethyl group or a formyl group, each R⁵denotes a methyl group.

M denotes two hydrogen atoms bonded to two pyrrole nitrogen atoms or anion of a transition metal belonging to the fourth to fifth periods inthe Periodic Table such as chromium, manganese, iron, cobalt orruthenium. As the transition metal, iron or cobalt is preferred. In thecase where the porphyrin compound of the present invention is used as anartificial oxygen carrier adapted for use in the living body, iron isparticularly preferred.

X⁻ denotes a halogen ion that is present in the case where M denotes thetransition metal ion, i.e., where the porphyrin compound of the presentinvention is a metal complex.

Further, n which denotes the number of X is the number obtained bysubtracting 2 from the valency of the transition metal ion.

As apparent from the definitions of R⁴ and R⁵ given above, the porphyrincompound of the present invention includes the compound represented byformula (A1) given below and the compound represented by formula (A2)given below:

where R¹-R³, X⁻, and n are as defined above, and R⁴ denotes a hydrogenatom, a vinyl group, an ethyl group, a 1-methoxyethyl group, a1-bromoethyl group or a formyl group.

where R¹-R³, X⁻, and n are as defined above, and R⁵ denotes a hydrogenatom, a vinyl group, an ethyl group, a 1-methoxyethyl group, a1-bromoethyl group or a formyl group.

The porphyrin compound of the present invention can be synthesized byvarious methods. For example, the compound can be synthesized byreacting a porphyrin represented by formula (1) given below with ahistidine derivative represented by formula (2) given below:

where R⁴ and R⁵ are as defined above;

where R² and R³ are as defined above.

In carrying out the reaction, the porphyrin represented by formula (1)may be used in the form of, for example, a disodium salt. Also, thehistidine derivative represented by formula (2) may be used in the formof a hydrohalide salt. The porphyrin represented by formula (1)includes, for example, protoporphyrin IX, meso-porphyrin IX, anddeuteroporphyrin IX.

Also, the histidine derivative represented by formula (2) can beprepared according to an ordinary method by subjecting histidine and theα-amino acid: H₂N(R)CHCOOH noted above to amide forming reaction betweenthe amino group of histidine and the carboxyl group of the α-amino acid,followed by modifying the carboxyl group of histidine. In other words,the histidine derivative can be obtained by the reaction between thehistidine after reaction with α-amino acid and an alcohol: R⁰OH, whereR⁰ denotes a C₁-C₁₈ alkyl group to esterify the histidine (where R³denotes a C₁-C₁₈ alkyl group), or with an amine: R⁰NH₂, where R⁰ denotesa C₁-C₁₈ alkyl group to amidate the histidine (where R³ denotes a C₁-C₁₈alkylamino group), according to an ordinary method. Alternatively, whereR³ denotes a peptide, the histidine after reaction with the α-amino acidis successively subjected to the reaction with the α-amino acid:H₂N(R)CHCOOH according to an ordinary method, and the C-terminal isconverted into a benzyl ester or an amino ester according to an ordinarymethod, as required. The histidine derivative represented by formula (2)is commercially available.

More specifically, the porphyrin represented by formula (1) is dissolvedin a suitable organic solvent such as pyridine or dimethylformamide(DMF), followed by adding an activating agent for activating thecarboxyl group of the porphyrin represented by formula (1), for example,(benzotriazole-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphoric acid (BOP), to the resultant solution, and themixture thus obtained is stirred for, e.g., 10 minutes to one hour. Theactivating agent such as BOP can be used in an amount of at least 2moles relative to one mole of porphyrin represented by formula (1) inthe case where the histidine derivative represented by formula (2) isreacted with one of the two carboxyl groups of porphyrin, and the othercarboxyl group is modified as described herein later. On the other hand,activating agent such as BOP can be used in an amount of 1.0-2.0 molesrelative to one mole of the porphyrin represented by formula (1) in thecase where the histidine derivative represented by formula (2) isreacted with one of the two carboxyl groups of the porphyrin and theother carboxyl group is not modified.

Then, the histidine derivative represented by formula (2), which isdissolved in a suitable organic solvent such as DMF or pyridine, isslowly added by using, for example, a dropping funnel to the reactionmixture after the stirring noted above. The histidine derivative can beused in an amount of 0.5 to 1.0 mole relative to one mole of theporphyrin represented by formula (1). After completion of the addition,the reaction mixture is stirred at room temperature for, e.g., 1 to 12hours under the condition that the light is shielded so as to carry outthe desired reaction. A porphyrin compound represented by formula (3)given below can be obtained by this reaction:

Then, where the carboxyl group that is not bonded to histidine informula (3) given above is modified, the porphyrin compound representedby formula (3) is reacted with an alcohol: R⁰OH, where R⁰ denotes aC₁-C₁₈ alkyl group, to esterify the carboxyl group (where R¹ is a C₁-C₁₈alkyl oxy group), or with an amine: R⁰NH₂, where R⁰ denotes a C₁-C₁₈alkyl group, to amidate the carboxyl group (where R¹ is a C₁-C₁₈ alkylamino group). Also, where R¹ denotes the peptide, the histidine afterthe reaction with the α-amino acid is successively reacted with theα-amino acid: H₂N(R)CHCOOH so as to convert the C-terminal into a benzylester or an amino ester by an ordinary method, as required. As a result,a porphyrin compound represented by formula (4) given below is obtainedin which R¹ denotes an atomic group other than the hydroxyl group:

In order to introduce a transition metal M into the porphyrin compoundrepresented by formula (3) or formula (4), it is possible to employ thegeneral method described in, for example, The Porphyrin, 1978, compiledby D. Dolphin and published by Academic Press Inc. In general, an iron(III) complex is obtained in the case of an iron complex, or a cobalt(II) complex is obtained in the case of a cobalt complex.

Among the porphyrin metal complexes described above, the iron (III)complex can be reduced into the iron (II) complex by using a suitablereducing agent such as sodium dithionite or ascorbic acid according toan ordinary method so as to develop an oxygen binding activity.

As apparent from the definition of formula (1), the porphyrin compoundof the present invention comprises a metal free porphyrin compound inwhich M denotes two hydrogen atoms, and a porphyrin metal complex inwhich M denotes a transition metal. Where the central metal M is in +2in the latter metal complex, the imidazole group of the histidinederivative covalently bonded to the propionic acid residue bonded to theporphyrin ring is coordinated with the central metal M as a proximalbase. As a result, the porphyrin metal complex of the present inventionthe +2 central metal M can exhibit an oxygen binding capability byitself so as to make it unnecessary to add from the outside an imidazolederivative as an axial base. Where the porphyrin metal complex comprisesiron or cobalt as the central metal M, the porphyrin metal complex canbe encapsulated in a phospholipid bi-layer vesicle by an ordinary method(see Japanese Patent Disclosure No. 8-301873 referred to previously), orcan be enclosed in a phospholipid-encapsulated fat emulsion (seeJapanese Patent Disclosure No. 6-184156). The porphyrin metal complexcan also be included in albumins such as a bovine serum albumin, a humanserum albumin, a recombinant human serum albumin, and a polymer albumin(see T. Komatsu, et al., Bioconjugate Chem., 13, 387 (2002)). Among thesystem thus obtained, the porphyrin iron or cobalt complex of thepresent invention is capable of promptly forming a stable oxygenatedcomplex upon contact with oxygen even in an aqueous system. Also, theporphyrin metal complex of the present invention is capable of adsorbingand desorbing oxygen in accordance with the oxygen partial pressure. Thebinding and dissociating of oxygen can be reversibly effectedrepeatedly. It should be noted that, in the porphyrin metal complex ofthe present invention, a derivative of histidine, which is one of thenatural amino acids, is used as a proximal base, and a porphyrin havingthe meso-position not modified is used as the porphyrin. It follows thatthe porphyrin metal complex of the present invention can provide auseful artificial oxygen carrier that is highly excellent in theadaptability to the living body.

It is expected for the porphyrin metal complex of the present inventionto be utilized as an artificial oxygen carrier in, for example, asubstitute for a transfusing blood, a blood diluent before the surgicaloperation, a replenishing solution in an external circulating circuitsuch as an artificial lung, a perfusion solution of a transplantedinternal organs, an oxygen supply solution into the ischemia portion, atreating agent of a chronic amenia, a perfusion solution for a liquidventilation, a sensitizing agent for curing cancer, and a culturesolution for a regenerated tissue cell. It is also expected for theporphyrin metal complex of the present invention to be utilized in arare blood type patient, a patient who denies blood transfusion forreligious reasons, and an animal therapy.

In addition, where the porphyrin compound of the present invention is inthe form of a complex of an ion of a metal belonging to, for example,the fourth period of the Periodic Table, the complex can be utilized asa catalyst in an oxygen reducing reaction, an oxygen oxidizing reactionor an oxygen addition reaction. In other words, the porphyrin metalcomplex of the present invention can be used as, particularly, anadsorbing-desorbing agent of an oxygen gas, as a redox catalyst, as acatalyst for an oxygen oxidizing reaction, and as a catalyst for anoxygen addition reaction in addition to the use as an artificial oxygencarrier.

In addition to oxygen, a gas capable of coordination with the centralmetal M, such as carbon monoxide, nitrogen monoxide or nitrogen dioxidecan form a coordinated complex with the porphyrin metal complex of thepresent invention. It follows that the porphyrin metal complex of thepresent invention can be used as an adsorbent of such a gas.

The present invention will now be described with reference to Exampleswhich follow. However, the present invention should not be limited tothe following Examples.

EXAMPLE 1

Protoporphyrin IX (400 mg (0.71 mmol)) was dissolved in pyridine (40mL), and the resultant solution was stirred at room temperature for 10minutes. Then, BOP (840 mg (1.9 mmol)) was added to the solution, andthe solution was further stirred for 10 minutes. Further, a solution ofglycyl-L-histidine methyl ester dihydrochloride (129 mg (0.57 mmol)) inDMF (15 mL) was slowly added dropwise into the resultant solution byusing a dropping funnel. The reaction mixture was stirred for 2.5 hoursat room temperature under a light-shielded condition, followed by addingethanol (4.2 mL (71 mmol)) to the reaction mixture, and the mixture wasstirred for 18 hours. The resultant reaction solution (15 to 20 mL) wasadded dropwise into ice water (1 L) and, then, centrifugally separated(7,000 g, 30 minutes), followed by filtering the precipitated materialwith a G4 glass filter and subsequently dissolving the filtrate in achloroform/methanol mixed solvent. After the solvent was removed under areduced pressure, the residue was fractionated with a silica gel column(chloroform/methanol=10/1 (v/v). After the solvent was removed under areduced pressure from the obtained fraction, the residue was furtherfractionated with a silica gel column (silica gel-60,chloroform/methanol=15/1 (v/v)). The fraction thus obtained was driedunder vacuum to give 100 mg of the desired porphyrin compound:3,8-divinyl-2,7,12,18-tetramethyl-13-(2-((N-glycyl-(O-methyl)histidine)carbamoyl)ethyl)-17-((ethoxycarbonyl)ethyl)porphyrin(yield of 20%).

<Analytical Result>

Thin layer chromatography (MERCK silica gel plate,chloroform/methanol=10/1 (v/v); Rf: 0.42 (monospot)

FAB mass spectrum: 785 [M−H⁺]

Infrared ray absorption spectrum (cm⁻¹): 1635 (ν_(C═O) (amide)); 1725(ν_(C═O) (ester))

Visible light absorption spectrum (chloroform): λ_(max): 625; 577; 541;505; 405 nm

¹H-NMR (d-DMSO, TMS standard); δ(ppm): −4.6 (s, 2H, inner-NH; 2.7-2.9(m, 2H, Im-CH₂—); 3.0-3.5 (m, 18H, por-CH₃, —CH₂—CH₂ —CO—NH—, —CH₂—CH₂—COO—CH₂—CH₃); 3.6 (s, 2H, —CONH—CH₂ —CONH—); 3.8 (s, 3H, —OCH₃);4.0-4.3 (d, 4H, por-CH₂—); 4.3-4.5 (m, 1H, α-CH); 6.0-6.4 (m, 4H,vinyl=CH₂); 7.4 (s, 1H, imidazole ring H); 8.0-8.3 (m, 5H, vinyl-CH,imidazole ring H); 9.8-10 (m, 4H, meso position-H). “Im” given aboveindicates imidazole, and “por” indicates porphyrin; the same appliesbelow.

EXAMPLE 2

The porphyrin compound obtained in Example 1 (50 mg (64 μmol)) was addedto a mixed solution of anhydrous DMF (20 mL) and 2,6-lutidine (37.1 μL(1.13 mmol)), and the result solution was deaerated with argon for 20minutes. Then, ferric chloride tetrahydrate (89 mg (1.13 mmol)) wasadded to the resultant solution at 60□ and the mixture was stirred for 4hours under an argon gas atmosphere. The resultant reaction solution wasadded dropwise into ice water (1 L), followed by adding potassium iodide(5 g), and subsequently subjecting to a centrifugal separation (5000 g,20 minutes). Then, the precipitated material was filtered with a G4glass filter, followed by dissolving the filtrate in achloroform/methanol mixed solvent. After the solvent was removed under areduced pressure, the residue was fractionated with a silica gel column(chloroform/methanol=5/1 (v/v)). The fraction thus obtained was driedunder vacuum to give 33.3 mg of the desired porphyrin iron complex:[3,8-divinyl-2,7,12,18-tetramethyl-13-(2-((N-glycyl-(O-methyl)histidine)carbamoyl)ethyl]-17-((ethoxycarbonyl)ethyl)porphyrinato]iron (III) iodide (yield of 62%).

<Analytical Result>

Thin layer chromatography (MERCK silica gel plate,chloroform/methanol=10/1 (v/v); Rf: 0.5 (monospot)

FAB mass spectrum: 838 [M−I⁻]

Infrared ray absorption spectrum (cm⁻¹): 1660 (ν_(C═O) (amide)); 1734(ν_(C═O) (ester))

Visible light absorption spectrum (chloroform): λ_(max): 637; 508; 388nm

EXAMPLE 3

Meso-porphyrin IX (500 mg (0.78 mmol)) was dissolved in pyridine (40mL), and the resultant solution was stirred at room temperature for 10minutes. Then, BOP (930 mg (2.1 mmol)) was added to the resultantsolution, followed by further stirring the solution for 10 minutes.After a solution of alanyl-L-histidine decyl ester dihydrochloride (221mg (0.63 mmol)) in pyridine (15 mL) was slowly added dropwise into theresultant solution by using a dropping funnel, the solution was stirredat room temperature for 6 hours under a light-shielded condition. Afterthe solvent was removed under a reduced pressure, the residue wasextracted with chloroform, and washed twice with hydrochloric acid (pH5) and twice with water, followed by drying the extracted material overanhydrous sodium sulfate. The extracted material was filtered, and thesolvent was removed under a reduced pressure. Further, the residue wasfractionated with a silica gel column (chloroform/methanol=20/1 (v/v)).The fraction thus obtained was dried under vacuum to give 146 mg of thedesired porphyrin compound:3,8-diethyl-2,7,12,18-tetramethyl-13-(2-((N-alanyl-L-(O-decyl)histidine)carbamoyl)ethyl)-17-(carboxyethyl)porphyrinhydrochloride (yield of 20%).

<Analytical Result>

Thin layer chromatography (MERCK silica gel plate,chloroform/methanol=20/1 (v/v); Rf: 0.4 (monospot)

FAB mass spectrum: 939.5 [M−H⁺]

Infrared ray absorption spectrum (cm⁻¹): 1635 (ν_(C═O) (amide)); 1705(ν_(C═O) (carboxylic acid))

Visible light absorption spectrum (chloroform): λ_(max): 626; 576; 540;504; 404 nm

¹H-NMR (CDCl₃, TMS standard); δ(ppm): −4.6 (s, 2H, inner-NH; 0.96 (t,3H, —CH₂CH₂CH₃ ); 1.3-1.6 (m, 19H, —CH(CH₃ )—, —OCH₂(CH₂ ) ₈ CH₃); 2.0(m, 6H, por-CH₂CH₃ ): 2.7-2.9 (m, 2H, Im-CH₂—); 3.0-3.5 (m, 18H,por-CH₃, CH₂—CH₂ —CO—NH, —CH₂—CH₂ —COOH); 4.0 (m, 2H, —C(═O)O—CH₂ —);4.1-4.3 (m, 4H, por-CH₂ —CH₂—); 4.4 (d, 4H, por-CH₂ —CH₃); 4.6-4.7 (m,2H, α-CH); 7.4 (s, 1H, imidazole ring H); 8.0 (m, 1H, imidazole ring H);9.8-10 (m, 4H, meso position-H).

EXAMPLE 4

The porphyrin compound obtained in Example 3 (50 mg (53 μmol)) was addedto a mixed solution of anhydrous DMF (20 mL) and 2,6-lutidine (31 μL(0.93 mmol)), and an argon gas was passed through the resultant solutionfor 20 minutes. Further, ferric chloride tetrahydrate (73 mg (0.93mmol)) was added to the solution, followed by stirring the resultantmixture at 60□ for 4 hours under an argon gas atmosphere. After thesolvent was removed under a reduced pressure, the residue was extractedwith chloroform, followed by washing the extract several times with purewater and subsequently drying the extract over anhydrous sodium sulfate.The extract was filtered. After the solvent was removed under a reducedpressure, the residue was fractionated with a silica gel column(chloroform/methanol=20/1 (v/v). The fraction thus obtained was driedunder vacuum to give 30 mg of the desired porphyrin iron complex:[3,8-diethyl-2,7,12,18-tetramethyl-13-(2-((N-alanyl-L-(O-decyl)histidine)carbamoyl)ethyl)-17-(carboxyethyl)porphyrinato]iron (III) chloride (yield of 60%).

<Analytical Result>

Thin layer chromatography (MERCK silica gel plate,chloroform/methanol=20/1 (v/v); Rf: 0.37 (monospot)

FAB mass spectrum: 958 [M−Cl⁻]

Infrared ray absorption spectrum (cm⁻¹): 1635 (ν_(C═O) (amide)); 1705(ν_(C═O) (carboxylic acid))

Visible light absorption spectrum (chloroform): λ_(max): 636; 507; 387nm

EXAMPLE 5

Deuteroporphyrin IX dihydrochloride (400 mg (0.69 mmol)) was dissolvedin pyridine (50 mL), and the resultant solution was stirred at roomtemperature for 10 minutes. Then, BOP (816 mg (1.8 mmol)) was added tothe resultant solution, followed by further stirring the solution for 10minutes. After a solution of valyl-L-histidinyl leucyl-leucine methylester dihydrochloride (3741 mg (0.55 mmol)) in pyridine (15 mL) wasslowly added dropwise into the resultant solution by using a droppingfunnel, the solution was stirred at room temperature for 6 hours under alight-shielded condition. Further, hexylamine (9.1 mL (69 mmol)) wasadded dropwise to the solution, followed by further stirring thesolution for 15 hours. After the solvent was removed from the reactionmixture under a reduced pressure, the residue was extracted withchloroform, and washed with water, followed by drying the extractedmaterial over anhydrous sodium sulfate. The extracted material wasfiltered, and the solvent was removed under a reduced pressure. Theresidue was fractionated with a silica gel column (chloroform/diethylether=20/1 (v/v)). The fraction thus obtained was dried under vacuum togive 130 mg of the desired porphyrin compound:2,7,12,18-tetramethyl-13-(2-(hexylcarbamoyl)ethyl)-17-(2-((N-valyl-L-luhistidyl-leucyl-leucyl-(O-methyl)leucine)carbamoy)ethyl)porphyrin(yield of 15%).

<Analytical Result>

Thin layer chromatography (MERCK silica gel plate, chloroform/diethylether=20/1 (v/v); Rf: 0.4 (monospot)

FAB mass spectrum: 1257 [M−H⁺]

Infrared ray absorption spectrum (cm⁻¹): 1635 (ν_(C═O) (amide)))

Visible light absorption spectrum (chloroform): λ_(max): 627; 577; 541;505; 405 nm

¹H-NMR (CDCl₃, TMS standard); δ(ppm): −4.6 (s, 2H, inner-NH; 1.0 (m,27H, —CHCH₃ )CH₃ , —NH(CH₂)₉(CH₃ ); 1.3-1.5 (16H, m, —NH(CH₂)(CH₂ ) ⁸CH₃); 1.7-2.2 (m, 10H, —CH(CH₃)CH₃, —CH₂ CH(CH₃)CH₃; 2.7-2.9 (m, 2H,Im-CH₂—); 3.0-3.5 (m, 18H, por-CH₃, —CH₂—CH₂ —CO—NH—, —NH—CH₂ (CH₂)₈CH₃;3.7 (s, 3H, —OCH₃); 4.1-4.3 (m, 4H, por, —CH₂—); 4.3-4.5 (m, 5H, α-CH);7.4 (s, 1H, imidazole ring H); 8.0 (m, 1H, imidazole ring H); 9.8-10 (m,6H, por-H).

EXAMPLE 6

The porphyrin compound obtained in Example 5 (100 mg (80 μmol)) wasadded to a mixed solution of anhydrous DMF (30 mL) and 2,6-lutidine (46μL (1.40 mmol)), and the resultant solution was stirred by passing anargon gas into the solution. Further, ferric chloride tetrahydrate (109mg (1.40 mmol)) was added to the solution, followed by stirring theresultant mixture at 60□ for 5 hours under an argon gas atmosphere.After the solvent was removed under a reduced pressure, the residue wasextracted with chloroform, followed by washing the extract several timeswith purified water and subsequently drying the extract over anhydroussodium sulfate. The extract was filtered. After the solvent was removedunder a reduced pressure, the residue was fractionated with a silica gelcolumn (chloroform/diethyl ether=20/1 (v/v)). The fraction thus obtainedwas dried under vacuum to give 64 mg of the desired porphyrin ironcomplex:[2,7,12,18-tetramethyl-13-(2-(hexylcarbamoyl)ethyl)-17-(2-((N-valyl-L-histidyl-leucyl-leucyl-(O-methyl)leucine)carbamoyl)porphyrinato]iron (III) chloride (yield of 60%).

<Analytical Result>

Thin layer chromatography (MERCK silica gel plate, chloroform/diethylether=20/1 (v/v); Rf: 0.38 (monospot)

FAB mass spectrum: 1312 [M−Cl⁻]

Infrared ray absorption spectrum (cm⁻¹): 1660 (ν_(C═O) (amide))

Visible light absorption spectrum (chloroform): λ_(max): 637; 508; 388nm

EXAMPLE 7

After nitrogen substitution, 30 μM DMF solution (3 mL) of the porphyriniron complex obtained in Example 2 was mixed with a methanol solution ofsodium dithionite-18-crown-6-ether complex, and the mixture was stirredfor 10 minutes so as to reduce the central iron of the porphyrin ironcomplex into the divalent state. As a result, a DMF solution of[3,8-divinyl-2,7,12,18-tetramethyl-13-(2-(N-glycyl-(O-methyl)histidine)carbamoyl)ethyl]-17-((ethoxycarbonyl)ethyl)porphyrinato] iron (II) iodide was obtained. The visible lightabsorption spectrum of the solution showed λ_(max) at 442; 545; and 565nm, supporting that the porphyrin iron complex corresponds to the5-coordinated deoxy type in which one imidazole molecule is coordinatedwith the central iron.

EXAMPLE 8

The[3,8-divinyl-2,7,12,18-tetramethyl-13-(2-(N-glycyl-(O-methyl)histidine)carbamoyl)ethyl)-17-((ethoxycarbonyl)ethyl)porphyrinato]iron (II) iodide obtained in Example 7 was included into the human serumalbumin in accordance with the method disclosed in Japanese PatentDisclosure No. 8-301873, preparing a porphyrin iron complex. Then, adispersion of the porphyrin iron complex-albumin inclusion compound in aphosphate buffer (porphyrin iron (II) complex: 20 μM; serum albumin: 20μM) was put in a cell made of quartz for a spectral measurement, and thecell was hermetically sealed under a nitrogen gas atmosphere. Thevisible light absorption spectrum of the dispersion showed λ_(max) at423; 539; and 569 nm, supporting that the included porphyrin iron (II)complex forms an Fe(II) high spin 5-coordinated complex having a singleintramolecular axial base coordinated therein. When oxygen wasintroduced into the dispersion, λ_(max) of the visible light absorptionspectrum was changed to 421; 542; and 561 nm, clearly supporting that anoxygenated complex was formed. When a nitrogen gas was blown into thedispersion of the oxygenated complex, the visible light absorptionspectrum was reversibly changed from the oxygenation type spectrum intothe deoxy-type spectrum, supporting that the adsorption-desorption ofoxygen is reversibly brought about. Incidentally, it was possible toperform consecutively the adsorption-desorption of oxygen by alternatelyrepeating the oxygen blow and the nitrogen blow. Also, the half-lifeperiod of the oxygenated complex was 90 to 120 minutes at 25□, and thusis clearly more stable than the oxygen complex of the porphyrin ironcomplex-albumin inclusion compound reported in the prior art.

As described above, the present invention provides a porphyrin compoundcapable of forming an oxygenated complex having a further improvedstability. The albumin inclusion body of the porphyrin compound canfunction as an artificial oxygen carrier.

1. A compound represented by formula (A):

where R1 denotes a C1-C18 alkyloxy group or a C1-C18 alkylamino group;R2 denotes a residual group after removal of an amino group and acarboxyl group from an a-amino acid selected from the group consistingof glycine, alanine, valine, leucine and isoleucine; R3 denotes a C1-C18alkyloxy group, a C1-C18 alkylamino group, or a peptide having 2-6α-amino acids selected from the group consisting of glycine, alanine,valine, leucine and isoleucine and having a methoxy group at theC-terminal; each R4 and each R5 denote either a methyl group, or ahydrogen atom, a vinyl group, an ethyl group, a 1-methoxyethyl group, a1-bromoethyl group or a formyl group, wherein, where each R4 denotes amethyl group, each R5 denotes a hydrogen atom, a vinyl group, an ethylgroup, a 1-methoxyethyl group, 1-bromoethyl group or a formyl group, andwhere each R4 denotes a hydrogen atom, a vinyl group, an ethyl group, a1-methoxyethyl group, a 1-bromoethyl group or a formyl group, each R5denotes a methyl group; M denotes two hydrogen atoms bonded to the twopyrrole nitrogen atoms or an ion of a transition metal belonging to thefourth to fifth periods in the Periodic Table; X— denotes a halogen ionthat is present where M denotes the transition metal ion; and n whichdenotes the number of X is the number obtained by subtracting 2 from thevalency of the transition metal ion.
 2. The compound according to claim1, wherein each R4 denotes a hydrogen atom, a vinyl group, an ethylgroup, a 1-methoxy ethyl group, a 1-bromo ethyl group or a formyl group,and each R5 denotes a methyl group.
 3. The compound according to claim1, wherein each R4 denotes a methyl group, and each R5 denotes a vinylgroup, an ethyl group, a 1-methoxy ethyl group, a 1-bromo ethyl group ora formyl group.
 4. The compound according to claim 1, wherein M denotesFe or Co.
 5. The compound according to claim 4, wherein Fe is divalentor trivalent.
 6. The compound according to claim 4, wherein Co isdivalent.